Substrate binding and catalytic mechanism of class B β-lactamases: a molecular modelling study

Increased resistance to β-lactam antibiotics is mainly due to β-lactamases whose production by pathogenic bacteria makes their broad activity spectrum especially frightening. X-ray structures of several zinc β-lactamases have revealed the coordination of the two metal ions, but their mode of action remains unclear. Geometry optimisation of stable complexes along the reaction pathway of benzylpenicillin hydrolysis highlighted a proton shuttle occurring from D120 of the Bacillus cereus β-lactamase to the β-lactam nitrogen via Zn2 which is central to the network. First, the Zn1 ion has a structural role maintaining Zn-bound waters, WAT1 and WAT2, either directly or through the Zn1 tetrahedrally coordinated histidine ligands. The Zn2 ion has a more catalytic role, stabilising the tetrahedral intermediate, accepting the β-lactam nitrogen atom as a ligand. The role of Zn2 and the flexibility in the coordination geometry of both Zn ions is of crucial importance for catalysis. Received 14 August 2001; received after revision 19 October 2001; accepted 30 October 2001     

Specific localization of the catalytic subunits of protein kinase CK2 at the centrosomes

The protein kinase CK2 holoenzyme is composed of two regulatory β subunits and two catalytic α or α' subunits. Although experimental evidence for involvement of the enzyme in the regulation of cell proliferation is accumulating, the exact mechanism of its action is still unclear. The subcellular localization of the enzyme may be a key to its function. We have recently shown that the CK2 holoenzyme is tightly associated with the Golgi complex and the endoplasmic reticulum. Centrosomes, which organize spindle formation during the cell cycle and microtubule cytoskeleton formation and, thereby, the location and orientation of different organelles in the cell, are in close vicinity to the Golgi complex. Because several kinases and phosphatases have been described to regulate the functions of the centrosome, we analysed the association of CK2 with these organelles. Using biochemical cell fractionation and coimmunoprecipitation, we never found the holoenzyme but only the catalytic asubunits associated with the centrosome. These data were confirmed by immunoelectron microscopy. Thus, the present data point to a particular role of the catalytic α and α' subunit of protein kinase CK2, which may be different from their roles in the holoenzyme. Received 2 August 2002; received after revision 2 October 2002; accepted 22 October 2002 RID="*" ID="*"Corresponding author.     

β-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome

Staphylococci have two mechanisms for resistance to β-lactam antibiotics. One is the production of β-lactamases, enzymes that hydrolytically destroy β-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by β-lactam antibiotics. Strains of S. aureus exhibiting either β-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism. Received 2 April 2005; received after revision 15 July 2005; accepted 25 July 2005     

ERKs are the point of divergence of PKA and PKC activation by PTHrP in human skin fibroblasts

Parathyroid hormone-related peptide (PTHrP) receptors, coupled to trimeric G proteins, operate in most target cells through at least three different transduction routes: Gαs-mediated stimulation of adenylylcyclase (AC), Gαq-mediated activation of phospholipase Cβ (PLC) and mitogen-activated protein kinase (MAPK) activation. In this study we investigated the relative role of different pathways in human skin fibroblast prolifera-tion. Using chemical inhibitors and activators of signal transduction, we demonstrated that: (i) AC/cAMP and PLC/1,4,5 inositol triphosphate/diacylglycerol second-messenger systems are simultaneously activated following PTHrP binding to its receptors; (ii) the mitogenic response to PTHrP derives from a balance between two counteracting pathways – an activating route mediated by protein kinase C (PKC) and an inhibitory route mediated by protein kinase A (PKA); (iii) PTHrP mitogenic effects are largely dependent on MAPKs, whose activity can be modulate d by both PKA and PKC. Our results indicate that MAPKs are common targets of both transduction routes and, at the same time, their point of divergence in mediating PTHrP dual and opposite mitogenic effects. Received 2 August 2002; received after revision 10 September 2002; accepted 18 October 2002 RID="*" ID="*"Corresponding author.     

Structural features of prions explored by sequence analysis. II. A PrP<Superscript>Sc</Superscript> model

Prion diseases are neurodegenerative disorders associated with a conformational conversion of the prion PrP protein, in which the β-strand content increases and that of the α helix decreases. However, the structure of the pathogenous form PrP^Sc, occurring after conformational conversion of the normal cellular form PrP^C, is not yet known. From sequence analysis, we have previously proposed that helix H2 of the prion PrP^C structure might be a key region for this structural conversion. More recently, we identified the TATA box-binding protein fold as a putative scaffold that may locally satisfy the predicted secondary-structure organisation of PrP^Sc. In the present analysis, we detail the schematic construction of PrP^Sc monomeric and dimeric models, based on this hypothesis. These models are globally compatible with available data and therefore may provide further insights into the structurally and functionally elusive PrP protein. Some comments are also devoted to a comparison of the yeast Ure2p prion and animal prions. Received 29 July 2002; received after revision 24 October 2002; accepted 24 October 2002 RID="*" ID="*"Corresponding author.     

Glycoconjugates of the intestinal goblet cells of four cyprinids

The aim of this work was to show differences in the terminal and subterminal sugar composition of carbohydrate chains of glycoconjugates produced by the goblet cells of the intestines of four cyprinids. We analysed intestines of two herbivorous species – sneep and grass carp – and two omnivorous ones – chub and common carp. We compared four intestinal regions of every studied species. In every region, the presence of neutral and acidic glycoconjugates was confirmed. The smallest amount of acidic glycoconjugates was present in the second region of sneep intestine. Sulphated glycoconjugates were absent in the third and fourth region of chub intestine. Lectin histochemistry provided evidence for the presence of β-D-galactose, α-N-acetylgalactosamine, β-N-acetylglucosamine and sialic acids. Additionally, the occurrence of α-L-fucose in the goblet cells of chub, grass carp and sneep was confirmed. We tried to correlate the patern of glycoconjugate glycosylation with feeding habits of the studied fishes. Received 1 July 2002; received after revision 8 August 2002; accepted 19 August 2002 RID="*" ID="*"Corresponding author.     

Oligo-1,6-glucosidase and neopullulanase enzyme subfamilies from the alpha-amylase family defined by the fifth conserved sequence region

The α-amylase enzyme family is the largest family of glycoside hydrolases. It contains almost 30 different enzyme specificities covering hydrolases, transferases and isomerases. Some of the enzyme specificities from the family are closely related, others less so. This study, based on the analysis of 79 amino acid sequences, postulates two subfamilies in the framework of the α-amylase family: the oligo-1,6-glucosidase subfamily and the neopullulanase subfamily. The specific sequence in the fifth conserved sequence region of the family served as the basis for defining the subfamilies: QpDln for the oligo-1,6-glucosidase subfamily and MPKln for the neopullulanase subfamily. This conserved sequence region is proposed to be the selection marker that enables one to distinguish between the two subfamilies. The 'intermediary' sequence MPDLN can be characteristic of the so-called intermediary group with a mixed enzyme specificity of α-amylase, cyclomaltodextrinase and neopullulanase. The evolutionary trees clearly supported the proposed definition of the two subfamilies. Received 12 July 2002; received after revision 28 August 2002; accepted 24 September 2002 RID="*" ID="*"Corresponding author.     

Role of glutamic acid decarboxylase in the pathogenesis of type 1 diabetes

Glutamic acid decarboxylase (GAD) is considered to be one of the strongest candidate autoantigens involved in triggering β-cell-specific autoimmunity. The majority of recent onset type 1 diabetes patients and prediabetic subjects have anti-GAD antibodies in their sera, as do nonobese diabetic (NOD) mice, one of the best animal models for human type 1 diabetes. Immunization of young NOD mice with GAD results in the prevention or delay of the disease as a result of tolerizing autoreactive T cells. Autoimmune diabetes can also be prevented by the suppression of GAD expression in antisense GAD trans genic mice backcrossed with NOD mice for seven generations. These results support the hypothesis that GAD plays an important role in the development of T-cell-mediated autoimmune diabetes. However, there is some controversy regarding the role of GAD in the pathogenesis of diabetes. Whether GAD truly plays a key role in the initiation of this disease remains to be determined. The examination of the development of insulitis and diabetes in β-cell-specific GAD knockout NOD mice will answer this remaining question. Received 12 April 2002; received after revision 24 May 2002; accepted 27 May 2002 RID="*" ID="*"Corresponding author.     

Ectoprotein kinase-mediated phosphorylation of FAT/CD36 regulates palmitate uptake by human platelets

Glycoprotein IV (FAT/CD36) has been shown to be phosphorylated by a cAMP-dependent, platelet membrane-bound ectokinase. In this study, we demonstrate that ectophosphorylation of FAT/CD36 regulates initial palmitate uptake. This is the first time that short-term regulation of the activity of a long-chain fatty acid carrier could be shown. Phosphorylation of FAT/CD36 was paralleled by a significant decrease in initial palmitate uptake by morphologically and functionally intact platelets. Maximum inhibition of palmitate uptake was achieved at 0.5 nM extracellular ATP, being significantly decreased to 72% compared to the control. Inhibition of palmitate uptake was abolished by co-incubation with the specific protein kinase A inhibitor peptide PKI or with β,γ-methylene-ATP, and was reversible upon addition of alkaline phosphatase. An extracellular ATP concentration above 5 μM completely prevented the ectophosphorylation-mediated inhibition of palmitate uptake. We conclude that FAT/CD36-mediated palmitate uptake by human platelets is short-term regulated via cAMP-dependent ectophosphorylation of FAT/CD36. Received 18 July 2002; received after revision 29 August 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Thyroid hormone controls carnitine status through modifications of γ-butyrobetaine hydroxylase activity and gene expression

The carnitine system plays a key role in β-oxidation of long-chain fatty acids by permitting their transport into the mitochondrial matrix. The effects of hypothyroidism and hyperthyroidism were studied on γ-butyrobetaine hydroxylase (BBH), the enzyme responsible for carnitine biosynthesis in the rat. In rat liver, BBH activity was decreased in the hypothyroid state and increased in hyperthyroid animals. The modifications in BBH activity correlated with changes in the enzyme Vmax values. These changes were shown to be related to hepatic BBH mRNA abundance. Thyroid hormones are known to interact with lipid metabolism, in particular by increasing long-chain fatty acid oxidation through activation of carnitine-dependent fatty acid import into mitochondria. Our study showed that thyroid hormones also increased carnitine bioavailability. Received 23 October 2001; received after revision 11 January 2002; accepted 15 January 2002     

Opposite actions of testosterone and progesterone on UCP1 mRNA expression in cultured brown adipocytes

The brown adipose tissue (BAT) thermogenic response to diet-induced obesity and cold has been found to be gender dependent. In the present work, we aimed to investigate the effects of the main physiological male and female sex hormones, i.e. testosterone, progesterone and 17-β-estradiol, on the expression of uncoupling protein 1 (UCP1) – the main mediator of BAT thermogenesis – and on UCP2 and lipid accumulation in rodent brown adipocytes differentiated in culture. Testosterone-treated cells showed fewer and smaller lipid droplets than control cells and a dose-dependent inhibition of UCP1 mRNA expression, under adrenergic stimulation by norepinephrine (NE). These effects were reverted by the androgen receptor antagonist flutamide, suggesting they are dependent, at least in part, on the androgen receptor. Progesterone- and 17-β-estradiol-treated cells showed more and larger lipid droplets and progesterone stimulated NE-induced UCP1 mRNA expression at the lower concentration tested, but not at higher concentrations, suggesting that for brown adipocytes, this hormone is dose dependent. 17-β-Estradiol did not have any remarkable effect either on UCP1 or UCP2 mRNA expression. Interestingly, the specific progesterone receptor antagonist RU486 induced UCP1 and UCP2 mRNAs, including UCP1 mRNA expression in non-NE-treated brown adipocytes, suggesting a profound effect of this anti-progestagen on brown adipocyte thermogenic capacity. Thus, are conclude that testosterone, 17-β-estradiol, progesterone and RU486 have distinct actions on brown adipocytes, thus modulating UCP1 and UCP2 mRNA expression and/or lipid accumulation, and that sex hormones are factors that may explain in part the gender-dependent BAT thermogenic response. Received 24 June 2002; received after revision 20 August 2002; accepted 26 August 2002 RID="*" ID="*"Corresponding author.     

"Add-on" domains of<Emphasis Type="Italic"> Drosophila</Emphasis> β1,4-<Emphasis Type="Italic">N</Emphasis>-acetylgalactosaminyltransferase B in the stem region and its pilot protein

The glycolipid specific Drosophila melanogaster β1,4-N-acetylgalactosaminyltransferase B (β4GalNAcTB) depends on a zinc finger DHHC protein family member named GalNAcTB pilot (GABPI) for activity and translocation to the Golgi. The six-membrane spanning protein actually lacks the cysteine in the cytoplasmic DHHC motif, displaying DHHS instead. Here we show that the whole conserved region around the DHHS sequence, which is essential for palmitoylation in DHHC proteins, is not required for GABPI to interact with β4GalNAcTB. In contrast, the two luminal loops between transmembrane domain 3–4 and 5–6 contain conserved amino acids, which are crucial for activity. Besides the dependence on GABPI, β4GalNAcTB requires its exceptional short stem region for activity. A few hydrophobic amino acids positioned close to the transmembrane domain are essential for the interaction with GABPI. Along with its catalytic domain, β4GalNAcTB, thus, requires an area in its own stem region and two small luminal loops of GABPI as "add-on" domains. Moreover, some inactive GABPI mutants could be rescued by fusion with β4GalNAcTB, indicating their importance in direct GABPI-β4GalNAcTB interaction.     

Molecular mechanisms of thrombin function

The discovery of thrombin as a Na⁺-dependent allosteric enzyme has revealed a novel strategy for regulating protease activity and specificity. The allosteric nature of this enzyme influences all its physiologically important interactions and rationalizes a large body of structural and functional information. For the first time, a coherent mechanistic framework is available for understanding how thrombin interacts with fibrinogen, thrombomodulin and protein C, and how Na⁺ binding influences the specificity sites of the enzyme. This information can be used for engineering thrombin mutants with selective specificity towards protein C and for the rational design of potent active site inhibitors. Thrombin also serves as a paradigm for allosteric proteases. Elucidation of the molecular basis of the Na⁺-dependent allosteric regulation of catalytic activity, based on the residue present at position 225, provides unprecedented insights into the function and evolution of serine proteases. This mechanism represents one of the simplest and most important structure-function correlations ever reported for enzymes in general. All vitamin K-dependent proteases and some complement factors are subject to the Na⁺-dependent regulation discovered for thrombin. Na⁺ is therefore a key factor in the activation of zymogens in the coagulation and complement systems.     

New endo-beta-1,4-glucanases from the parabasalian symbionts,Pseudotrichonympha grassii and Holomastigotoides mirabile of Coptotermes termites

An endo-β-1,4-glucanase (EG) was purified from the hindgut of an Australian mound-building termite, Coptotermes lacteus. The hindgut extract had a peak separate from those for extracts obtained from the salivary glands and the midgut based on sephacryl S-200 gel chromatography, and also demonstrated an origin different from the endogenous EGs of the termite itself. The recovery was further purified by SDS-PAGE, and its N-terminal amino acid sequence analyzed. This showed high homology to EGs from glycoside hydrolase family (GHF) 7. PCR-based cloning methods were applied to the hindgut contents of C. lacteus and individual protozoan symbionts from C. formosanus. cDNAs encoding putative EGs homologous to GHF7 members were then identified. The functionality of one of the putative proteins was confirmed by its expression in Escherichia coli. Received 18 September 2002; accepted 20 September 2002 RID="*" ID="*"Corresponding author.     

Isoforms of soluble alpha-tubulin in oocytes and brain of the frog (genus Rana): changes during oocyte maturation

Rana oocytes have previously been shown to contain much more soluble tubulin than does the brain, suggesting different assembly and disassembly dynamics of frog oocyte tubulin compared to that in brain. By using centrifugation, SDS-PAGE, two-dimensional gel electrophoresis and Western blots, probed with anti-α-tubulin monoclonal antibodies, polymorphic α-tubulins (isoforms) were compared in brains and follicle-enclosed oocytes of northern (Rana pipiens) and southern (R. berlandieri) frogs. Oocyte tubulin in both species had isoforms with greater ranges of isoelectric point (pI) than those of brain tubulins; in particular, the oocyte tubulin pIs ranged further into the acidic region of the isoelectric-focusing gels than corresponding brain tubulin. This difference may, in part, be responsible for the previously reported assembly differences between oocyte tubulin (undetectable assembly) and brain tubulin (high assembly). Isoforms of α-tubulin with relat ively acidic pI were more abundant in northern frog brain and oocyte soluble extracts than in analogous extracts from southern frogs. Furthermore, additional acidic α-tubulin isoforms were found in progesterone-treated oocytes (i.e., eggs), indicating increased heterogeneity of acidic a-tubulin isoforms during oocyte meiotic maturation. Among northern frog oocyte soluble components fractionated on Superose-6b columns, tubulin complexes with apparent molecular mass of about 1800 kDa were found to contain acidic α-tubulin isoforms while the putative oligomeric tubulins with an apparent molecular mass of about 250 kDa contained an additional relatively basic α-tubulin isoform. The acidic α-tubulin isoforms, therefore, are proposed to be associated with cold-adaptable cells of brain and oocytes, and may also be involved in stabilization of large soluble tubulin complexes in oocytes of the northern frog. Received 1 October 2002; accepted 9 October 2002 RID="*" ID="*"Corresponding author.     

Morphofunctional changes in the mitochondrial subpopulations of conceptus tissues during the placentation process

To establish the role of mitochondrial subpopulations in the mitochondrial maturation process, we studied morphological and functional changes in the mitochondria of different mammalian conceptus tissues during the organogenic and the placentation processes. Mitochondrial subpopulations of three different conceptus tissues, embryo and visceral yolk sac placenta on gestational days 11, 12 and 13 and placenta on days 12 and 13, were examined morphologically by transmission electron microscopy. Cytochrome oxidase activity and protein levels were also measured in each mitochondrial subpopulation. The results indicate two different mitochondrial subpopulation profiles: a homogeneous one, which corresponds to immature mitochondria, and a heterogeneous one, which represents the mature mitochondria. The three tissues studied show different morphologic and metabolic patterns of mitochondrial maturation during the placentation process, rendering them suitable as experimental models to establish the p ossible relationship between mitochondrial maturation and the mitochondrial subpopulations. Received 5 August 2002; received after revision 23 September 2002; accepted 8 October 2002 RID="*" ID="*"Corresponding author.     

β-Lactamases as models for protein-folding studies

This review traces some of the key features of the folding of β-lactamases and their relevance to the way proteins fold in general. Studies on the enzymes have highlighted the nature and role of equilibrium and transient condensed states. The kinetics of folding are multiphasic, and when monitored by acrylamide quenching of the tryptophan fluorescence, an early phase provides evidence for the transient accumulation of a nonnative intermediate involving burial of tryptophan in a nonpolar environment. Intermediate phases can be understood in terms of progressive folding of different parts of the molecule. The later, slow phases are associated with proline isomerization in the TEM-1 enzyme and, in its P167T mutant form, with isomerization from trans to cis of the E166 T167 peptide bond. Coupled with kinetic and X-ray crystallographic studies of the β-lactamase from Staphylococcus aureus and its D179Q mutant, it appears that the final stage of folding is that of collapse and packing of the Ω-loop on to the main body of the protein.     

Cellulose synthesis: mutational analysis and genomic perspectives using Arabidopsis thaliana

Cellulose microfibrils containing crystalline β-1,4-glucan provide the major structural framework in higher-plant cell walls. Genetic analyses of Arabidopsis thaliana now link specific genes to plant cellulose production just as was achieved some years earlier with bacteria. Cellulose-deficient mutants have defects in several members of one family within a complex glycosyltransferase superfamily and in one member of a small family of membrane-bound endo-1,4-β-glucanases. The mutants also accumulate a readily extractable β-1,4-glucan that has short chains which, in at least one case, are lipid linked. Cellulose could be made by direct extension of the glucan chain by the glycosyltransferase or, as the mutant suggests, by an indirect route which makes lipid-linked oligosaccharides. Models discussed incorporate the known enzymes and lipo-glucan and raise the possibility that different CesA glycosyltransferases may catalyse different steps. Received 5 January 2001; received after revision 25 April 2001; accepted 25 April 2001     

Melatonin regulation of antioxidant enzyme gene expression

Antioxidant enzymes (AOEs) are part of the primary cellular defense against free radicals induced by toxins and/or spontaneously formed in cells. Melatonin (MLT) has received much attention in recent years due to its direct free radical scavenging and antioxidant properties. In the present work we report that MLT, at physiological serum concentrations (≈ 1 nM), increases the mRNA of both superoxide dismutases (SODs) and glutathione peroxidase (GPx) in two neuronal cell lines. The MLT effect on both SODs and GPx mRNA was mediated by a de novo synthesized protein. MLT alters mRNA stability for Cu-Zn SOD and GPx. Experiments with a short time treatment (pulse action) of MLT suggest that the regulation of AOE gene expression is likely to be receptor mediated, because 1-h treatment with MLT results in the same response as a 24-h treatment. Received 18 June 2002; received after revision 5 August 2002; accepted 27 August 2002 RID="*" ID="*"Corresponding author.     

Immunomodulatory properties of cystatins

Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. Because these cysteine proteases exist in all living organisms and because they are involved in various biological and pathological processes, the control of these protease functions by cystatins is of cardinal importance. Cystatins are found in mammals but cystatin-like molecules are also present in mammals and parasites. In the immune system, cystatins modulate cathepsin activities and antigen presentation. They also induce tumor necrosis factor α and interleukin 10 synthesis, and they stimulate nitric oxide production by interferon γ-activated murine macrophages. In turn, nitric oxide has inhibitory activity on cysteine proteases, especially those from parasitic protozoa. Cystatins isolated from parasitic nematodes also have immunomodulatory activities that are distinguishable from those induced by lipopolysacharide-like molecules from endosymbiotic bacteria. On the whole, cystatins and cystatin-like molecules belong to a new category of immunomodulatory molecules. Doubtless increasing data will improve our knowledge of this property, leading to practical applications in immunotherapy. Received 11 April 2002; accepted 18 April 2002 RID="*" ID="*"Corresponding author.